The present invention relates to the grafts in the oculistics and more particularly the production in vitro of laminae of human epithelium corneae containing stem cells from cultured limbar stem cells to be grafted directly to patients.
As known, stem cells of epithelium corneae are confined to the layer between cornea and conjunctiva, so-called limbus, and allow the upper layers to be continuously renewed.
In the severe ocular burns the loss of corneal tissue and the total or subtotal depletion of the limbar cells causes the missing epithelium to be replaced by the conjunctival cells forming naturally an opaque layer that causes the loss of sight.
The grafts of tissue portions formed of limbar cells allows the functionality of cornea to be recovered, the blindness due to the destructive lesion of the limbar-corneal epithelium to be overcome, and the perforating keratoplasty to be successfully carried out even in most complex cases where the number of successful results by using the known methods of the state of art is extremely small.
It is known that epithelial cells are difficult to be prepared and kept in vitro as they are fragile and hard to handle so that the methods used hitherto to produce tissues in vitro are little effective.
The present invention seeks to select effectively the limbar cells to be cultivated in vitro and to improve the method of cultivating the same on a fibrin substrate suitably modified to obtain reconstructed laminae of epithelium corneae ready to use in grafts. The human anterior ocular surface is covered with two highly specialized structures: conjunctive and cornea. The function of cornea is to refract light and to direct it to the visual perception region of retina and is responsible for focusing the images along with crystalline. The conjunctive covers the remaining bulbi oculi portion and performs an important function in keeping the normal homeostasis of the ocular surface. Although the conjunctival and limbar-corneal tissues have the typical characteristics of the epithelia, they are formed by two genotipically different cell types, hereafter referred to as conjunctival keratinocytes and limbar-corneal keratinocytes, respectively. In particular, epithelium corneae is renewed constantly and continuously because of the stem cells confined in the deepest transitional layer between cornea and conjunctive, so-called limbus, where the transient amplifying cells (TA cells) forming the epithelium corneae are originated. Epithelium corneae is completely renewed every 9-12 months.
The reconstruction in vitro of the epithelial tissue aiming at repairing and/or replacing those natural, for example, as a result of severe burns is widely supported with documents. Cells are cultivated in vitro on suitable biological substrates helping the cell adherence and proliferation as well as allowing the proper architecture of the epithelial tissue to be obtained. One of the most used substrate is fibrin which is available even in commercial form as TISSUCOL (by Baxter-Immuno, Vienna, Austria), a solution of two components: fibrinogen and thrombin. Such composition prepared according to the protocol provided by the manufacturer, however, is not suitable to be used in cultures of limbar-corneal keratinocytes as it is not transparent and elastic enough.
The present invention overcomes the problems relative to the preparation of homogeneous, steady cultivated corneal stem cells. Such cells growing on a suitably modified fibrin substrate originate in vitro laminae of epithelium corneae having all of the characteristics of the human cornea such as size, thickness and transparency. In addition, such method allows such elastic laminae of epithelium corneae to adhere perfectly to the ocular surface without having to cause it to bend during the development in vitro (as described by the same inventor in his Italian Patent No. RM9200408). Therefore, the laminae of human epithelium corneae produced in vitro according to the present invention are ready to use and adapted to any patient.
One object of the present invention is a method by which it is possible to prepare in laboratory laminae of human epithelium corneae to be used in grafts. Such method requires a number of steps to obtain homogeneous cell cultures containing stem cells and capable of easily forming in vitro laminae of human epithelium corneae ready to use because of the modified fibrin substrate.
Further features and advantages of the invention will be more readily apparent from the following detailed description of an embodiment with reference to the accompanying drawings.